Limited induction of polyfunctional lung-resident memory T cells against SARS-CoV-2 by mRNA vaccination compared to infection

Resident memory T cells (TRM) present at the respiratory tract may be essential to enhance early SARS-CoV-2 viral clearance, thus limiting viral infection and disease. While long-term antigen-specific TRM are detectable beyond 11 months in the lung of convalescent COVID-19 patients, it is unknown if mRNA vaccination encoding for the SARS-CoV-2 S-protein can induce this frontline protection. Here we show that the frequency of CD4+ T cells secreting IFNγ in response to S-peptides is variable but overall similar in the lung of mRNA-vaccinated patients compared to convalescent-infected patients. However, in vaccinated patients, lung responses present less frequently a TRM phenotype compared to convalescent infected individuals and polyfunctional CD107a+ IFNγ+ TRM are virtually absent in vaccinated patients. These data indicate that mRNA vaccination induces specific T cell responses to SARS-CoV-2 in the lung parenchyma, although to a limited extend. It remains to be determined whether these vaccine-induced responses contribute to overall COVID-19 control.

Patients undergoing lung resection for various reasons at the Vall d'Hebron University Hospital were recruited through the Thoracic Surgery Service and invited to participate. Adult patients included in this study were 24 years old or older and were assigned to one of the following groups: I.) SARS-CoV-2 uninfected unvaccinated individuals (Ctrl, n=5), II.) unvaccinated longterm SARS-CoV-2 convalescent individuals (Inf, n=9, convalescent for~4-12 months), III.) uninfected and long-term two-or three-dose vaccinated individuals (LT, n=10,~4-10 months after the last vaccine), and IV.) uninfected and short-term threeor four-dose vaccinated individuals (ST, n=6, 1.3-1.8 months after the last vaccine). Patient information is summarized in Supplementary Table 1 and a schematic summary is shown in Figure 1a.
To assign patients to their group, clinical history was consulted to confirm or rule out previous infection with SARS-CoV-2. Moreover, plasma samples of most patients were analysed for the presence of total Ig against N-protein and IgG against Sprotein, which discriminated Control patients (negative for N and S IgG), Convalescent Infection patients (positive for N and S) and vaccinated uninfected patients (negative for N and positive for S).
Recruitment of patients relied on their need to undergo lung resection for various reasons. Thus, recruitment and availability of patients primarily relied on surgical planification. Additionally, recruitment of patients to our predefined study groups was dependent on their vaccination and/or SARS-CoV-2 infection status as described in the Methods section. Surgeries were performed at the Vall d'Hebron University Hospital and patients were recruited through the Thoracic Surgery Service and invited to participate. It is unlikely that this selection created a bias in our results as we predefined group conditions and included patients meeting these requirements. As additional controls for certain analyses requiring only blood, healthy volunteers recently boosted with a fourth vaccine dose (within 1 week) were recruited. Lastly, no other selection criteria were applied to all groups present in this study.
This study was performed in accordance with the Declaration of Helsinki and approved by the corresponding Institutional Review Board (PR(AG)212/2020) of the Vall d'Hebron University Hospital (HUVH), Barcelona, Spain. Written informed consent was provided by all patients recruited to this study.
No sample-size calculations were performed. Next to tissue availability, sample size was determined to be adequate based on the magnitude and consistency of measurable differences between groups.
No data were excluded after selection of patients meeting the requirements for assignment to one of the four study groups. Study groups were: I.) SARS-CoV-2 uninfected unvaccinated individuals, II.) unvaccinated long-term SARS-CoV-2 convalescent individuals (convalescent for 4-12 months), III.) uninfected and long-term two-or three-dose vaccinated individuals (~4-10 months after the last vaccine), and IV.) uninfected and short-term three-or four-dose vaccinated individuals (1.3-1.8 months after the last vaccine).
Given the uniqueness of the paired blood and lung samples, experiments to measure SARS-CoV-2 specific responses in blood and lung samples were optimized and replicated before starting this study. Individual lung and blood samples were not replicated due to sample limitation. After optimization, transwell experiments were performed with available blood and lung samples. A minimum of nine Transwell replicates were used for each experiment for blood and lung samples separately. All replication attempts were successful.
Allocation to experimental groups was not random, as our research aim did not allow for random allocation. Patients were first selected on meeting predefined criteria of our study groups based on their recorded clinical history (i.e. history of proven SARS-CoV-2 infection and mRNA vaccination status). Predefined study group criteria were: I.) SARS-CoV-2 uninfected unvaccinated individuals, II.) unvaccinated long-term SARS-CoV-2 convalescent individuals (convalescent for~4-12 months), III.) uninfected and long-term two-or three-dose vaccinated individuals (~4-10 months after the last vaccine), and IV.) uninfected and short-term three-or four-dose vaccinated individuals (1.3-1.8 months after the last vaccine). After the first selection based on clinical records, the status of patients in the vaccine groups was confirmed by serological testing: vaccinated patients with presence of total Ig antibodies against the N protein were excluded from the study.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. All antibodies used in this study are commercially available and were commercially validated. They have been used according to manufacturer data sheets available at the manufacturer´s website. In house titration of antibodies was performed to determine the optimal dilution for our experiments.

Materials
According to the manufacturer's website, anti-CD107a (clone H4A3) antibody (BD Biosciences, Cat#555802) has been routinely tested on the fixed and permeabilized Jurkat cells by flow cytometry with Cytofix/Cytoperm (Cat. No. 554714) for fixation and permeabilization. This antibody was previously validated in Chen JW et al., 1988, J Biol Chem. According to the manufacturer's website, anti-CD103 (clone Ber-ACT8) antibody (Biolegend, Cat# 350204) is quality control tested by immunofluorescent staining with flow cytometric analysis. Flow cytometric analysis of CD103 expression in human peripheral blood mononuclear cells is provided on the website. According to the manufacturer's website, anti-CD69 (clone FN50) antibody (BD Biosciences, Cat#562617) is suitable for flow cytometry. Flow cytometric analysis of CD69 expression by stimulated peripheral blood mononuclear cells is provided on the website. According to the manufacturer's website, anti-CD40 (clone HB14) antibody (Biolegend, Cat#313017) is quality control tested by immunofluorescent staining with flow cytometric analysis. Flow cytometric analysis of CD40 expression in human peripheral blood lymphocytes is provided on the website. According to the manufacturer's website, anti-CD8 (clone RPA-T8) antibody (BD Biosciences, Cat#561952) is routinely tested for flow cytometry. Flow cytometric analysis of CD8 expression on human peripheral blood lymphocytes is provided on the website. According to the manufacturer's website, anti-CD3 (clone UCHT1) antibody (BD Biosciences, Cat#563851) is routinely tested for flow cytometry. Flow cytometric analysis of CD3 expression on human peripheral blood lymphocytes is provided on the website. According to the manufacturer's website, anti-CD45 (clone HI30) antibody (BD Biosciences, Cat#564047) is routinely tested for flow cytometry. Flow cytometric analysis of CD45 expression on human peripheral blood lymphocytes is provided on the website. According to the manufacturer's website, anti-IL-4 (clone 8D4-8) antibody (eBioscience, Cat# 25-7049-82) is suitable for flow cytometry. Flow cytometric analysis of IL-4 expression on human peripheral cells is provided on the website. According to the manufacturer's website, anti-IL-10 (clone JES3-19F1) antibody (BD Biosciences, Cat# 559330) has been routinely